Sepsis and multiple organ dysfunction continue to be leading causes of morbidity and mortality in critically ill surgical patients. Cytokine mediators appear to be responsible for many clinical manifestations. Most studies of macrophage regulation utilize a single bacterial stimulus, although it is unlikely that patients are exposed to a single insult. The hypothesis here is that sequential endotoxin (LPS) stimulation would alter macrophage cytokine release and provide insights into the dysregulated cytokine secretion seen in sepsis. Using an in vitro murine macrophage model, the same stimulus (LPS) applied to the same cells 8-24 hours after pretreatment produce reprogrammed patterns of TNF and IL-1 release. The present studies are designed to systematically investigate the mechanisms by which pretreatment modulates subsequent LPS stimulated cytokine release. The secretion of both TNF and IL-1 are regulated via both pre and post transcriptional mechanisms. The proposal will examine the impact of pretreatment reprogramming on mRNA transcription, transcription rate, the stability of cytokine message and post translational protein processing and release. The pathways of LPS induced signal transduction will be compared with and without pretreatment using specific signal transduction agonists and antagonists. Alterations in signal transduction pathways will be compared to other well characterized activating stimuli such as phorbol esters, zymosan, and calcium ionophore. Systematic investigations of the role of possible LPS pretreatment induced alterations in LPS receptor binding, activation of protein kinases, and early gene activation will be performed.